Production of Olfactory Receptors and Nanosomes Using Yeast System for Bioelectronic Nose - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 125

Fig. 8.1 Calcium imaging on dissociated cells stimulated by a flow of diluted target odorant in
normal saline buffer at the indicated concentration during the periods in-between the dashed verti-
cal lines. OSNs 1-6 respond to various odorant concentrations by transitory intracellular calcium
pulses. OSNs 1 and 2, OSNs 3 and 4, OSNs 5 and 6 were shown to host OR1 , OR2 , and OR3 ,
respectively
Our lab followed yet another strategy, requiring several preliminary steps to in-
crease the chances of success [ 20 ]. Since the expression of most ORs is spatially
restricted in the olfactory mucosa, we first explored which of the 4 main turbinates
best responds to the target odorant by performing ElectroOlfactoGram (EOG) re-
cordings on rat olfactory mucosa [ 21 ]. Different doses of the target odorant were
applied in a vapor phase to potentially stimulate and locate different responding
neurons. The olfactory tissue from the best responding turbinates was dissected
and prepared to plate freshly dissociated cells as described [ 22 ]. We then combined
calcium imaging analysis of cells stimulated using the target odorant and single-cell
reverse transcription of collected cells, as previously described [ 23 , 24 ]. Figure 8.1
shows the calcium response profile of representative responding cells stimulat-
ed with increasing odorant concentrations. The whole cell content of individual
responsive neurons was collected and used for mRNA retrotranscription. Controls
corresponding to nonresponsive cells or culture medium were also prepared
in parallel. The sequence of the OR expressed in each neuron was amplified by
nested-PCR using 2 pairs of rat degenerate primers designed to match the high-
est proportion of olfactory receptors sequences. This design is not trivial because
of the large number of OR genes in a given species and of their highly variable
sequences, which prevents a total covering of the OR sequences, even using degen-
erate primers positioned over highly conserved sequences. Indeed, identities range